The invention, in some embodiments, relates to the field of molecular interactions, and particularly to methods and devices useful in producing local concentrations of protons, proton concentration gradients and desired proton concentration topographies in an environment (e.g., a solution, a gel, or the like) including an electrolyte. Some embodiments of the invention also relate to the field of analyte separation and isoelectric focusing. Some embodiments of the invention also relate to the field of data display.
Isoelectric focusing is an analytical technique for separating molecules in an analyte sample by taking advantage of the differing ionic properties of the molecules.
Isoelectric focusing is performed in a gel (usually of materials such as polyarylamide polyacrylamide, starch or agarose) having an immobilized proton concentration gradient, generally the proton concentration gradient changing from higher to lower pH in a given direction.
The analyte is loaded onto some location on the gel. The charge of each different molecule changes in response to the ambient proton concentration according to the acidity (pKa) of the various functional groups of the molecule.
An electric potential is applied parallel to the proton concentration gradient between an isoelectric focusing anode and isoelectric focusing cathode. Molecules having a net positive charge migrate through the gel towards the anode while molecules having a net negative charge migrate through the gel towards the cathode. The opposite, positively charge ions (cations) migrate towards the cathode and negatively charged ions (anions) migrate towards the anode.
As the molecules migrate, the ambient pH changes to reduce the net charge on the molecule until the molecule reaches an isoelectric point (pI) where, due to the ambient pH, the net charge on the molecule is zero so that the molecule stops migrating due to the electric potential. If a molecule “overshoots” the isolectric point, the molecule reverses direction.
In such a way, isoelectric focusing focuses molecules having the same pI into very narrow well-defined volumes of the gel.
Isoelectric focusing is exceptionally useful for the analysis of proteins as proteins are characterized by having many functional groups of different acidities.
Isoelectric focusing suffers from a number of disadvantages. To have sufficient resolution, it is often necessary to have a number of different gels having different proton concentration gradients spanning different ranges of proton concentration gradients, increasing costs and creating a logistical problem. Automated manipulation of fragile gels is difficult to implement. Gels having immobilized proton concentration gradients are generally expensive and may suffer from batch to batch reproducibility. Analysis of some analytes may suffer from a sieving effect as large proteins may have difficulty migrating through pores in the gels. Isoelectric focusing may be slow due to the slow migration of the analyte molecules.
It would be advantageous to be able to perform isoelectric focusing with fewer disadvantages of the methods known in the art.